Treatment of cardiovascular and related pathologies

ABSTRACT

Methods for treating cardiovascular and related diseases such as ischemia, ischemia reperfusion injuries, and myocardial ischemia, are described. The methods are directed to concurrently administering a compound such as pyridoxal-5′-phosphate, pyridoxamine, pyridoxal, or a 3-acylated pyridoxal analogue with a therapeutic cardiovascular compound.

This application is a divisional of U.S. application Ser. No.09/645,237, now U.S. Pat. No. 6,677,356, filed Aug. 24, 2000, whichclaims the benefit under 35 U.S.C. §119(e) of U.S. ProvisionalApplication No. 60/150,415, filed Aug. 24, 1999, the entire disclosuresof which are hereby incorporated by reference.

FIELD OF THE INVENTION

This invention relates to methods of treating cardiovascular and relateddiseases, such as hypertrophy, hypertension, congestive heart failure,ischemia, such as myocardial ischemia, ischemia reperfusion injuries invarious organs, arrhythmia, and myocardial infarction.

BACKGROUND

Heart failure is a pathophysiological condition in which the heart isunable to pump blood at a rate commensurate with the requirement of themetabolizing tissues or can do so only from an elevated filling pressure(increased load). Thus, the heart has a diminished ability to keep upwith its workload. Over time, this condition leads to excess fluidaccumulation, such as peripheral edema, and is referred to as congestiveheart failure.

When an excessive pressure or volume load is imposed on a ventricle,myocardial hypertrophy (i.e., enlargement of the heart muscle) developsas a compensatory mechanism. Hypertrophy permits the ventricle tosustain an increased load because the heart muscle can contract withgreater force. However, a ventricle subjected to an abnormally elevatedload for a prolonged period eventually fails to sustain an increasedload despite the presence of ventricular hypertrophy, and pump failuremay ultimately occur.

Heart failure can arise from any disease that affects the heart andinterferes with circulation. For example, a disease that increases theheart muscle's workload, such as hypertension, will eventually weakenthe force of the heart's contraction. Hypertension is a condition inwhich there is an increase in resistance to blood flow through thevascular system. This resistance leads to increases in systolic and/ordiastolic blood pressures. Hypertension places increased tension to theleft ventricular myocardium, causing it to stiffen and hypertrophy, andaccelerates the development of atherosclerosis in the coronary arteries.The combination of increased demand and lessened supply increases thelikelihood of myocardial ischemia leading to myocardial infarction,sudden death, arrhythmias, and congestive heart failure.

Ischemia is a condition in which an organ or a part of the body fails toreceive a sufficient blood supply. When an organ is deprived of itsblood supply, it is said to be hypoxic. An organ will become hypoxiceven when the blood supply temporarily ceases, such as during a surgicalprocedure or during temporary artery blockage. Ischemia initially leadsto a decrease in or loss of contractile activity. When the organaffected is the heart, this condition is known as myocardial ischemia,and myocardial ischemia initially leads to abnormal electrical activity.This may generate an arrhythmia. When myocardial ischemia is ofsufficient severity and duration, cell injury may progress to celldeath—i.e., myocardial infarction—and subsequently to heart failure,hypertrophy, or congestive heart failure.

When blood flow resumes to an organ after temporary cessation, this isknown as ischemic reperfusion of the organ. For example, reperfusion ofan ischemic myocardium may counter the effects of coronary occlusion, acondition that leads to myocardial ischemia. Ischemic reperfusion to themyocardium may lead to reperfusion arrhythmia or reperfusion injury. Theseverity of reperfusion injury is affected by numerous factors, such as,for example, duration of ischemia, severity of ischemia, and speed ofreperfusion. Conditions observed with ischemia reperfusion injuryinclude neutrophil infiltration, necrosis, and apoptosis.

Drug therapies, using known active ingredients such as vasodilators,angiotensin II receptor antagonists, angiotensin converting enzymeinhibitors, diuretics, antithrombolytic agents, β-adrenergic receptorantagonists, α-adrenergic receptor antagonists, calcium channelblockers, and the like, are available for treating heart failure andassociated diseases. Of course, any drug used for treatment may resultin side effects. For example, vasodilators may result in hypotension,myocardial infarction, and adverse immune response. Angiotensin IIreceptor antagonists and angiotensin converting enzyme inhibitors areoften associated with acute renal failure, fetopathic potential,proteinuria, hepatotoxicity, and glycosuria as side effects. Similarly,common side effects associated with calcium channel blockers includehypotension, peripheral edema, and pulmonary edema. β-Adrenergicreceptor antagonists and diuretics have been associated withincompatibility with nonsteroidal anti-inflammatory drugs in addition toimpotence, gout, and muscle cramps in the case of diuretics and inaddition to a decrease in left ventricular function and suddenwithdrawal syndrome in the case of β-adrenergic receptor antagonists.Moreover, side effects associated with α-adrenergic receptor antagonistsinclude thostatic hypotension, and side effects associated withantithrombolytic agents include excessive bleeding.

To address the side effects, the dosage of a drug may be reduced or theadministration of the drug may be abated and replaced with another drug.It would be desirable to administer a drug therapy with decreasedamounts of the active ingredient to reduce side effects but maintaineffectiveness.

SUMMARY OF THE INVENTION

The present invention provides methods for treating cardiovascular andrelated diseases, such as, for example, hypertrophy, hypertension,congestive heart failure, myocardial ischemia, ischemia reperfusioninjuries in an organ, arrhythmia, and myocardial infarction. Oneembodiment is directed to a method of treating cardiovascular disease ina mammal by concurrently administering to the mammal a therapeuticallyeffective amount of a combination of a compound suitable for use inmethods of the invention and a therapeutic cardiovascular compound.Therapeutic cardiovascular compounds suitable for use in methods of theinvention include an angiotensin converting enzyme inhibitor, anangiotensin II receptor antagonist, a calcium channel blocker, anantithrombolytic agent, a β-adrenergic receptor antagonist, avasodilator, a diuretic, an α-adrenergic receptor antagonist, anantioxidant, and a mixture thereof. In some embodiments, the therapeuticcardiovascular compound is PPADS.

Compounds suitable for use in the methods of the invention includepyridoxal-5′-phosphate, pyridoxamine, pyridoxal, 3-acylated pyridoxalanalogues, pharmaceutically acceptable acid addition salts thereof, andmixtures thereof.

In one embodiment, a 3-acylated pyridoxal analogue is a compound of theformula

In another embodiment, a 3-acylated pyridoxal analogue is a compound ofthe formula

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 is a graph showing the effect of P-5-P and aspirin, alone or incombination, on mortality in the rat model of coronary ligation.

FIG. 2 is a graph showing the effect of P-5-P and captopril, alone or incombination, on mortality in the rat model of coronary ligation.

FIG. 3 is a graph showing the effect of P-5-P and propranolol, alone orin combination, on mortality in the rat model of coronary ligation.

FIG. 4 is a graph showing the effect of P-5-P and verapamil, alone or incombination, on mortality in the rat model of coronary ligation.

FIG. 5 is a graph showing the effect of P-5-P and aspirin, alone or incombination, on scar weight in the rat model of coronary ligation.

FIG. 6 is a graph showing the effect of P-5-P and captopril, alone or incombination, on scar weight in the rat model of coronary ligation.

FIG. 7 is a graph showing the effect of P-5-P and propranolol, alone orin combination, on scar weight in the rat model of coronary ligation.

FIG. 8 is a graph showing the effect of P-5-P and verapamil, alone or incombination, on scar weight in the rat model of coronary ligation.

FIG. 9 is a graph showing the effect of P-5-P and aspirin, alone or incombination, on the rate of force of contraction (+dp/dt) in the ratmodel of coronary ligation.

FIG. 10 is a graph showing the effect of P-5-P and captopril, alone orin combination, on the rate of force of contraction (+dp/dt) in the ratmodel of coronary ligation.

FIG. 11 is a graph showing the effect of P-5-P and propranolol, alone orin combination, on the rate of force of contraction (+dp/dt) in the ratmodel of coronary ligation.

FIG. 12 is a graph showing the effect of P-5-P verapamil, alone or incombination, on the rate of force of contraction (+dp/dt) in the ratmodel of coronary ligation.

FIG. 13 is a graph showing the effect of P-5-P and aspirin, alone or incombination, on the rate of force of relaxation (−dp/dt) in the ratmodel of coronary ligation.

FIG. 14 is a graph showing the effect of P-5-P and captopril, alone orin combination, on the rate of force of relaxation (−dp/dt) in the ratmodel of coroary ligation.

FIG. 15 is a graph showing the effect of P-5-P and propranolol, alone orin combination, on the rate of force of relaxation (−dp/dt) in the ratmodel of coronary ligation.

FIG. 16 is a graph showing the effect of P-5-P and verapamil, alone orin combination, on the rate of force of relaxation (−dp/dt) in the ratmodel of coronary ligation.

FIG. 17 is a graph showing the effect of P-5-P and aspirin, alone or incombination, on left ventricular end diastolic pressure (LVEDP) in therat model of coronary ligation.

FIG. 18 is a graph showing the effect of P-5-P and captopril, alone orin combination, on left ventricular end diastolic pressure (LVEDP) inthe rat model of coronary ligation.

FIG. 19 is a graph showing the effect of P-5-P and propranolol, alone orin combination, on left ventricular end diastolic pressure (LVEDP) inthe rat model of coronary ligation.

FIG. 20 is a graph showing the effect of P-5-P and verapamil, alone orin combination, on left ventricular end diastolic pressure (LVEDP) inthe rat model of coronary ligation.

FIG. 21 is a graph showing the effect of P-5-P and aspirin, alone or incombination, on heart weight in the rat model of coronary ligation.

FIG. 22 is a graph showing the effect of P-5-P and captopril, alone orin combination, on heart weight in the rat model of coronary ligation.

FIG. 23 is a graph showing the effect of P-5-P propranolol, alone or incombination, on heart weight in the rat model of coronary ligation.

FIG. 24 is a graph showing the effect of P-5-P and verapamil, alone orin combination, on heart weight in the rat model of coronary ligation.

FIG. 25 is a graph showing the effect of P-5-P and aspirin, alone or incombination, on right ventricular weight in the rat model of coronaryligation.

FIG. 26 is a graph showing the effect of P-5-P and captopril, alone orin combination, on right ventricular weight in the rat model of coronaryligation.

FIG. 27 is a graph showing the effect of P-5-P and propranolol, alone orin combination, on right ventricular weight in the rat model of coronaryligation.

FIG. 28 is a graph showing the effect of P-5-P and verapamil, alone orin combination, on right ventricular weight in the rat model of coronaryligation.

FIG. 29A is a graph showing systolic blood pressure in rats from allpretreatment experiment groups at “0” day. “C” designates a controlgroup; “S” designates a sucrose diet induced diabetic group; “M”designates a group administered P-5-P alone; “Ca” designates a groupadministered captopril alone; “V” designates a group administeredverapamil alone; “M+Ca” designates a group administered P-5-P andcaptopril; “M+V” designates a group administered P-5-P and verapamil.

FIG. 29B is a graph showing the effect of pretreatment with P-5-P,captopril and verapamil on systolic blood pressure in rats whenadministered 1 week prior to sucrose diet induced diabetes. “C”, “S”,“M”, “Ca”, “V”, “M+Ca”, and “M+V” are designated as in FIG. 29A.

FIG. 30A is a graph showing systolic blood pressure in rats from allexperiment groups involved in same day treatment as sucrose feeding at“0” day. “C”, “S”, “M”, “Ca”, “V”, “M+Ca”, and “M+V” are designated asin FIG. 29A.

FIG. 30B is a graph showing the effect of administration of P-5-P,captopril and verapamil on systolic blood pressure in rats whenadministered the same day as sucrose feeding to induce diabetes. “C”,“S”, “M”, “Ca”, “V”, “M+Ca”, and “M+V” are designated as in FIG. 29A.

FIG. 31A is a graph showing systolic blood pressure in rats from allexperiment groups involved in treatment two weeks after sucrose feedingat “0” day. “C”, “S”, “M”, “Ca”, “V”, “M+Ca”, and “M+V” are designatedas in FIG. 29A.

FIG. 31B is a showing systolic blood pressure in rats from allexperiment groups involved in treatment two weeks after sucrose feedingat “0” day. “C”, “S”, “M”, “Ca”, “V”, “M+Ca”, and “M+V” are designatedas in FIG. 29A.

DESCRIPTION OF THE INVENTION

The present invention provides methods for treatment of cardiovascularand related diseases or conditions. Such cardiovascular and relateddiseases include hypertrophy, hypertension, congestive heart failure,ischemia, such as myocardial ischemia, ischemia reperfusion injury,arrhythmia, and myocardial infarction.

In accordance with the present invention, it has been found thatpyridoxal-5′-phosphate and its derivatives can be used concurrently withtherapeutic cardiovascular compounds in the treatment of theabove-identified diseases and conditions. “Treatment” and “treating” asused herein include preventing, inhibiting, and alleviatingcardiovascular diseases, related diseases, and related symptoms as wellas healing the ischemia-related conditions or symptoms thereof affectingmammalian organs and tissues. Treatment may be carried out byconcurrently administering a therapeutically effective amount of acombination of a compound suitable for use in methods of the inventionand a therapeutic cardiovascular compound.

A “therapeutically effective amount” as used herein includes aprophylactic amount, for example, an amount effective for preventing orprotecting against cardiovascular diseases, related diseases, andsymptoms thereof, and amounts effective for alleviating or healingcardiovascular diseases, related diseases, and symptoms thereof. Byadministering a compound suitable for use in methods of the inventionconcurrently with a therapeutic cardiovascular compound, the therapeuticcardiovascular compound may be administered in a dosage amount that isless than the dosage amount required when the therapeutic cardiovascularcompound is administered as a sole active ingredient. By administeringlower dosage amounts of the active ingredient, the side effectsassociated therewith should accordingly be reduced.

Compounds suitable for use in the methods of the invention includepyridoxal-5′-phosphate, pyridoxal, pyridoxamine, 3-acylated pyridoxalanalogues, pharmaceutically acceptable acid addition salts thereof, andmixtures thereof. 3-Acylated pyridoxal analogues provide for slowermetabolism to pyridoxal in vivo. For example, a suitable 3-acylatedanalogue of pyridoxal(2-methyl-3-hydroxy-4-formyl-5-hydroxymethylpyridine) is a compound ofthe formula I:

or a pharmaceutically acceptable acid addition salt thereof, wherein

R₁ is a straight or branched alkyl group, a straight or branched alkenylgroup, in which an alkyl or alkenyl group may be interrupted by anitrogen or oxygen atom; an alkoxy group; a dialkylamino group; or anunsubstituted or substituted aryl group.

The term “alkyl” group includes a straight or branched saturatedaliphatic hydrocarbon chain having from 1 to 8 carbon atoms, such as,for example, methyl, ethyl, propyl, isopropyl (1-methylethyl), butyl,tert-butyl (1,1-dimethylethyl), and the like.

The term “alkenyl” group includes an unsaturated aliphatic hydrocarbonchain having from 2 to 8 carbon atoms, such as, for example, ethenyl,1-propenyl, 2-propenyl, 1-butenyl, 2-methyl-1-propenyl, and the like.

The above alkyl or alkenyl groups may optionally be interrupted in thechain by a heteroatom, such as, for example, a nitrogen or oxygen atom,forming an alkylaminoalkyl or alkoxyalkyl group, for example,methylaminoethyl or methoxymethyl, and the like.

The term “alkoxy” group includes an alkyl group as defined above joinedto an oxygen atom having preferably from 1 to 4 carbon atoms in astraight or branched chain, such as, for example, methoxy, ethoxy,propoxy, isopropoxy (1-methylethoxy), butoxy, tert-butoxy(1,1-dimethylethoxy), and the like.

The term “dialkylamino” group includes two alkyl groups as defined abovejoined to a nitrogen atom, in which the alkyl group has preferably 1 to4 carbon atoms, such as, for example, dimethylamino, diethylamino,methylethylamino, methylpropylamino, diethylamino, and the like.

The term “aryl” group includes an aromatic hydrocarbon group, includingfused aromatic rings, such as, for example, phenyl and naphthyl. Suchgroups may be unsubstituted or substituted on the aromatic ring by, forexample, an alkyl group of 1 to 4 carbon atoms, an alkoxy group of 1 to4 carbon atoms, an amino group, a hydroxy group, or an acetyloxy group.

Preferred R₁ groups for compounds of formula I are toluyl or naphthyl.Such R₁ groups when joined with a carbonyl group form an acyl group

which preferred for compounds of formula I include toluoyl orβ-naphthoyl. Of the toluoyl group, the p-isomer is more preferred.

Examples of 3-acylated analogues of pyridoxal include, but are notlimited to, 2-methyl-3-toluoyloxy-4-formyl-5-hydroxymethylpyridine and2-methyl-βnaphthoyloxy-4-formyl-5-hydroxymethylpyridine.

Another suitable analogue is a 3-acylated analogue ofpyridoxal-4,5-aminal (1-secondaryamino-1,3-dihydro-7-hydroxy-6-methylfuro(3,4-c)pyridine) of the formulaII:

or a pharmaceutically acceptable acid addition salt thereof, wherein

R₁ is a straight or branched alkyl group, a straight or branched alkenylgroup, in which an alkyl or alkenyl group may be interrupted by anitrogen or oxygen atom; an alkoxy group; a dialkylamino group; or anunsubstituted or substituted aryl group; and

R₂ is a secondary amino group.

The terms “alkyl,” “alkenyl,” “alkoxy,” “dialkylamino,” and “aryl” areas defined above.

The term “secondary amino” group includes a group of the formula III:

derived from a secondary amine R₃R₄NH, in which R₃ and R₄ are eachindependently alkyl, alkenyl, cycloalkyl, aryl, or, when R₃ and R₄ aretaken together, may form a ring with the nitrogen atom and which mayoptionally be interrupted by a heteroatom, such as, for example, anitrogen or oxygen atom. The terms “alkyl,” “alkenyl,” and “aryl” areused as defined above in forming secondary amino groups such as, forexample, dimethylamino, methylethylamino, diethylamino, dialkylamino,phenylmethylamino, diphenylamino, and the like.

The term “cycloalkyl” refers to a saturated hydrocarbon having from 3 to8 carbon atoms, preferably 3 to 6 carbon atoms, such as, for example,cyclopropyl, cyclopentyl, cyclohexyl, and the like.

When R₃ and R₄ are taken together with the nitrogen atom, they may forma cyclic secondary amino group, such as, for example, piperidino, and,when interrupted with a heteroatom, includes, for example, piperazinoand morpholino.

Preferred R₁ groups for compounds of formula II include toluyl, e.g.,p-toluyl, naphthyl, tert-butyl, dimethylamino, acetylphenyl,hydroxyphenyl, or alkoxy, e.g., methoxy.

Such R₁ groups when joined with a carbonyl group form an acyl group

which preferred for compounds of formula II include toluoyl,β-naphthoyl, pivaloyl, dimethylcarbamoyl, acetylsalicyloyl, salicyloyl,or alkoxycarbonyl. A preferred secondary amino group may be morpholino.

Examples of 3-acylated analogues of pyridoxal-4,5-aminal include, butare not limited to,1-morpholino-1,3-dihydro-7-(p-toluoyloxy)-6-methylfuro(3,4-c)pyridine;1-morpholino-1,3-dihydro-7-(β-naphthoyloxy)-6-methylfuro(3,4-c)pyridine;1-morpholino-1,3-dihydro-7-pivaloyloxy-6-methylfuro(3,4-c)pyridine;1-morpholino-1,3-dihydro-7-carbamoyloxy-6-methylfuro(3,4-c)pyridine; and1-morpholino-1,3-dihydro-7-acetylsalicyloxy-6-methylfuro(3,4-c)pyridine.

The compounds of formula I may be prepared by reacting pyridoxalhydrochloride with an acyl halide in an aprotic solvent. A suitable acylgroup is

wherein R₁ is as defined above. A particularly suitable acyl halideincludes p-toluoyl chloride or β-naphthoyl chloride. A suitable aproticsolvent includes acetone, methylethylketone, and the like.

The compounds of formula II may be prepared by reacting 1-secondaryamino-1,3-dihydro-7-hydroxy-6-methylfuro(3,4-c)pyridine with an acylhalide in an aprotic solvent. An acyl group is

wherein R₁ is as defined above. A particularly suitable acyl halideincludes p-toluoyl chloride, β-naphthoyl chloride, trimethylacetylchloride, dimethylcarbamoyl chloride, and acetylsalicyloyl chloride. Aparticularly suitable secondary amino group includes morpholino.

The compound1-morpholino-1,3-dihydro-7-hydroxy-6-methylfuro(3,4-c)pyridine may beprepared by methods known in the art, for example, by reactingmorpholine and pyridoxal hydrochloride at a temperature of about 100° C.in a solvent. A suitable solvent includes, for example, toluene.Similarly, other secondary amines as defined for R₂ may be used asreactants to prepare the appropriate 1-secondary amino compounds.

The compounds of formula I may alternatively be prepared from thecompounds of formula II by reacting a compound of formula II with anaqueous acid, such as, for example, aqueous acetic acid.

One skilled in the art would recognize variations in the sequence andwould recognize variations in the appropriate reaction conditions fromthe analogous reactions shown or otherwise known that may beappropriately used in the above-described processes to make thecompounds of formulas I and II herein.

The products of the reactions described herein are isolated byconventional means such as extraction, distillation, chromatography, andthe like.

Pharmaceutically acceptable acid addition salts of compounds suitablefor use in methods of the invention include salts derived from nontoxicinorganic acids such as hydrochloric, nitric, phosphoric, sulfuric,hydrobromic, hydriodic, hydrofluoric, phosphorous, and the like, as wellas the salts derived from nontoxic organic acids, such as aliphaticmono- and dicarboxylic acids, phenyl-substituted alkanoic acids, hydroxyalkanoic acids, alkanedioic acids, aromatic acids, aliphatic andaromatic sulfonic acids, etc. Such salts thus include sulfate,pyrosulfate, bisulfate, sulfite, bisulfite, nitrate, phosphate,monohydrogenphosphate, dihydrogenphosphate, metaphosphate,pyrophosphate, chloride, bromide, iodide, acetate, trifluoroacetate,propionate, caprylate, isobutyrate, oxalate, malonate, succinate,suberate, sebacate, fumarate, maleate, mandelate, benzoate,chlorobenzoate, methylbenzoate, dinitrobenzoate, phthalate,benzenesulfonate, toluenesulfonate, phenylacetate, citrate, lactate,maleate, tartrate, methanesulfonate, and the like. Also contemplated aresalts of amino acids such as arginate and the like and gluconate,galacturonate N-methyl glutamine, etc., as disclosed for example byBerge et al. in their publication entitled Pharmaceutical Salts, in J.Pharmaceutical Science, 66: 1–19 (1977). Berge et al. outlined variouspotential useful salts along with their physicochemical studies, theirbioavailabilities, their pharmacological studies and their toxicologies.

The acid addition salts of the basic compounds are prepared bycontacting the free base form with a sufficient amount of the desiredacid to produce the salt in the conventional manner. The free base formmay be regenerated by contacting the salt form with a base and isolatingthe free base in the conventional manner. The free base forms differfrom their respective salt forms somewhat in certain physical propertiessuch as solubility in polar solvents, but otherwise the salts areequivalent to their respective free base for purposes of the presentinvention.

Methods of the invention include concurrently administeringpyridoxal-5′-phosphate, pyridoxamine, pyridoxal, a 3-acylated pyridoxalanalogue, a pharmaceutically acceptable acid addition salt thereof, or amixture thereof with a therapeutic cardiovascular compound to treathypertrophy, hypertension, congestive heart failure, ischemia, such asmyocardial ischemia, ischemia reperfusion injury, arrhythmia, ormyocardial infarction. Preferably, the cardiovascular disease treated ishypertrophy or congestive heart failure. Still preferably, thecardiovascular disease treated is arrhythmia. Also preferably, thecardiovascular disease treated is ischemia reperfusion injury.

Therapeutic cardiovascular compounds that may be concurrentlyadministered with a compound suitable for use in methods of theinvention include an angiotensin converting enzyme inhibitor, anangiotensin II receptor antagonist, a calcium channel blocker, anantithrombolytic agent, a β-adrenergic receptor antagonist, avasodilator, a diuretic, an α-adrenergic receptor antagonist, anantioxidant, and a mixture thereof. A compound suitable for use inmethods of the invention also may be concurrently administered withPPADS (pyridoxal phosphate-6-azophenyl-2′,4′-disulphonic acid), also atherapeutic cardiovascular compound, or with PPADS and another knowntherapeutic cardiovascular compound as already described. In a preferredembodiment, pyridoxal-5′-phosphate is concurrently administered withPPADS or with PPADS and another known therapeutic cardiovascularcompound, preferably an angiotensin converting enzyme inhibitor or anangiotensin II receptor antagonist.

Preferably, a therapeutic cardiovascular compound, which is concurrentlyadministered with pyridoxal-5′-phosphate, pyridoxamine, pyridoxal, a3-acylated pyridoxal analogue, a pharmaceutically acceptable acidaddition salt thereof, or a mixture thereof, is an angiotensinconverting enzyme inhibitor, an angiotensin II receptor antagonist, or adiuretic. Still preferably, the therapeutic cardiovascular compound isan α-adrenergic receptor antagonist. Also preferably, the therapeuticcardiovascular compound is a calcium channel blocker.

These therapeutic cardiovascular compounds are generally used to treatcardiovascular and related diseases as well as symptoms thereof. Askilled physician or veterinarian readily determines a subject who isexhibiting symptoms of any one or more of the diseases described aboveand makes the determination about which compound is generally suitablefor treating specific cardiovascular conditions and symptoms.

For example, myocardial ischemia may be treated by the administrationof, for example, angiotensin converting enzyme inhibitor, an angiotensinII receptor antagonist, a calcium channel blocker, an antithrombolyticagent, a β-adrenergic receptor antagonist, a diuretic, an α-adrenergicreceptor antagonist, or a mixture thereof. In some instances, congestiveheart failure may be treated by the administration of, for example,angiotensin converting enzyme inhibitor, an angiotensin II receptorantagonist, a calcium channel blocker, a vasodilator, a diuretic, or amixture thereof.

Myocardial infarction may be treated by the administration of, forexample, angiotensin converting enzyme inhibitor, a calcium channelblocker, an antithrombolytic agent, a β-adrenergic receptor antagonist,a diuretic, an α-adrenergic receptor antagonist, or a mixture thereof.

Hypertension may be treated by the administration of, for example,angiotensin converting enzyme inhibitor, a calcium channel blocker, aβ-adrenergic receptor antagonist, a vasodilator, a diuretic, anα-adrenergic receptor antagonist, or a mixture thereof.

Moreover, arrhythmia may be treated by the administration of, forexample, a calcium channel blocker, a β-adrenergic receptor antagonist,or a mixture thereof.

Antithrombolytic agents are used for reducing or removing blood clotsfrom arteries.

Hypertropy may be treated by the administration of, for example, anangiotensin converting enzyme inhibitor, an angiotensin II receptorantagonist, a calcium channel blocker, or a mixture thereof.

Ischemia reperfusion injury may be treated by the administration of, forexample, an angiotensin converting enzyme inhibitor, an angiotensin IIreceptor antagonist, a calcium channel blocker, or a mixture thereof.

Known angiotensin converting enzyme inhibitors include, for example,captopril, enalapril, lisinopril, benazapril, fosinopril, quinapril,ramipril, spirapril, imidapril, and moexipril.

Examples of known angiotensin II receptor antagonists include bothangiotensin I receptor subtype antagonists and angiotensin II receptorsubtype antagonists. Suitable angiotensin II receptor antagonistsinclude losartan and valsartan.

Suitable calcium channel blockers include, for example, verapamil,diltiazem, nicardipine, nifedipine, amlodipine, felodipine, nimodipine,and bepridil.

Antithrombolytic agents known in the art include antiplatelet agents,aspirin, and heparin.

Examples of known β-adrenergic receptor antagonists include atenolol,propranolol, timolol, and metoprolol.

Suitable vasodilators include, for example, hydralazine, nitroglycerin,and isosorbide dinitrate.

Suitable diuretics include, for example, furosemide, diuril, amiloride,and hydrodiuril.

Suitable α-adrenergic receptor antagonists include, for example,prazosin, doxazocin, and labetalol.

Suitable antioxidants include vitamin E, vitamin C, and isoflavones.

A compound suitable for use in methods of the invention and atherapeutic cardiovascular compound are administered concurrently.“Concurrent administration” and “concurrently administering” as usedherein includes administering a compound suitable for use in methods ofthe invention and a therapeutic cardiovascular compound in admixture,such as, for example, in a pharmaceutical composition or in solution, oras separate compounds, such as, for example, separate pharmaceuticalcompositions or solutions administered consecutively, simultaneously, orat different times but not so distant in time such that the compoundsuitable for use in methods of the invention and the therapeuticcardiovascular compound cannot interact and a lower dosage amount of theactive ingredient cannot be administered.

A physician or veterinarian of ordinary skill readily determines asubject who is exhibiting symptoms of any one or more of the diseasesdescribed above. Regardless of the route of administration selected, thecompound suitable for use in methods of the invention and thetherapeutic cardiovascular compound are formulated into pharmaceuticallyacceptable unit dosage forms by conventional methods known to thepharmaceutical art. An effective but nontoxic quantity of the compoundsuitable for use in methods of the invention and the therapeuticcardiovascular compound are employed in the treatment.

The compound suitable for use in methods of the invention and thetherapeutic cardiovascular compound may be concurrently administeredenterally and/or parenterally in admixture or separately. Parenteraladministration includes subcutaneous, intramuscular, intradermal,intramammary, intravenous, and other administrative methods known in theart. Enteral administration includes tablets, sustained release tablets,enteric coated tablets, capsules, sustained release capsules, entericcoated capsules, pills, powders, granules, solutions, and the like.

A pharmaceutical composition suitable for administration comprises apharmaceutically acceptable carrier and a compound suitable for use inmethods of the invention and/or a therapeutic cardiovascular compound.The pharmaceutical composition comprises a pharmaceutically acceptablecarrier and a compound suitable for use in methods of the invention,such as, for example, pyridoxal-5′-phosphate, pyridoxal, pyridoxamine, a3-acylated pyridoxal analogue, a pharmaceutically acceptable acidaddition salt thereof, and a mixture thereof. A pharmaceuticallyacceptable carrier includes, but is not limited to, physiologicalsaline, ringers, phosphate buffered saline, and other carriers known inthe art. Pharmaceutical compositions may also include stabilizers,antioxidants, colorants, and diluents. Pharmaceutically acceptablecarriers and additives are chosen such that side effects from thepharmaceutical compound are reduced or minimized and the performance ofthe compound is not canceled or inhibited to such an extent thattreatment is ineffective.

Methods of preparing pharmaceutical compositions containing apharmaceutically acceptable carrier and a compound suitable for use inmethods of the invention and/or a therapeutic cardiovascular compoundare known to those of skill in the art. All methods may include the stepof bringing the compound suitable for use in methods of the inventionand/or the therapeutic cardiovascular compound in association with thecarrier or additives. In general, the formulations are prepared byuniformly and intimately bringing the compound into association with aliquid carrier or a finely divided solid carrier or both, and then, ifnecessary, shaping the product into the desired unit dosage form.

The ordinarily skilled physician or veterinarian will readily determineand prescribe the therapeutically effective amount of the compound totreat the disease for which treatment is administered. In so proceeding,the physician or veterinarian could employ relatively low dosages atfirst, subsequently increasing the dose until a maximum response isobtained. Typically, the particular disease, the severity of thedisease, the compound to be administered, the route of administration,and the characteristics of the mammal to be treated, for example, age,sex, and weight, are considered in determining the effective amount toadminister. Administering a therapeutically effective amount of acompound suitable for use in methods of the invention to treatcardiovascular and related diseases as well as symptoms thereof istypically in a range of about 0.1–100 mg/kg of a patient's body weight,more preferably in the range of about 0.5–50 mg/kg of a patient's bodyweight per daily dose when administered alone. The compound suitable foruse in methods of the invention may be administered for periods of shortand long duration.

Therapeutically effective amounts of respective therapeuticcardiovascular compounds when administered as sole active ingredientsare known in the art and may be found in, for example, Physicians' DeskReference (53^(rd) ed., 1999).

When concurrently administering a compound suitable for use in methodsof the invention and a therapeutic cardiovascular compound, the compoundsuitable for use in methods of the invention is typically administeredin a range of about 0.1–100 mg/kg of a patient's body weight, preferably0.5–50 mg/kg of a patient's body weight, per daily dose, and thetherapeutic cardiovascular compound is administered in an amount lessthan the amount known in the art, which is administered when thetherapeutic cardiovascular compound is administered as the sole activeingredient. Typically, the therapeutic cardiovascular compound isadministered in an amount at least 5% less than the amount known in theart, which is administered when the therapeutic cardiovascular compoundis administered as the sole active ingredient.

A therapeutically effective amount of a compound suitable for use inmethods of the invention and a therapeutic cardiovascular compound fortreating cardiovascular and related diseases and symptoms thereof can beadministered prior to, concurrently with, or after the onset of thedisease or symptom. For example, a therapeutically effective amount of acompound suitable for use in methods of the invention and a therapeuticcardiovascular compound for treating ischemia reperfusion injury ormyocardial infarction can be administered before, during, or followingischemia (including during or following reperfusion), as well ascontinually for some period spanning from pre- to post-ischemia. Forexample, the compound suitable for use in methods of the invention and atherapeutic cardiovascular compound may be concurrently administeredprior to heart procedures, including bypass surgery, thrombolysis, andangioplasty, and prior to any other procedures that require blood flowbe interrupted and then resumed. Additionally, a compound suitable foruse in methods of the invention and a therapeutic cardiovascularcompound may be taken on a regular basis to protect against cellulardysfunction arising from arrhythmia and heart failure.

The invention is further elaborated by the representative examples asfollows. Such examples are not meant to be limiting.

EXAMPLES Example 1 Synthesis of morpholine pyridoxal-4,5-aminal(1-morpholino-1,3-dihydro-7-hydroxy-6-methylfuro(3,4-c)pyridine)

A mixture of morpholine (20 g) and toluene (100 mL) was stirred andheated using an oil bath set to 100° C. for 15 minutes. Pyridoxalhydrochloride (10 g) was then added and the reaction mixture was stirredat 100° C. for two hours. The reaction mixture was then concentrated bydistillation of the toluene and morpholine. The concentrated reactionmixture was washed three times by adding toluene (100 mL) and removingthe toluene by distillation. After washing, the residue was dissolved intoluene and filtered, and then hexane was added until precipitationbegan, at which time the reaction mixture was left overnight at roomtemperature. Crystals were collected and washed thoroughly with hexane.

Nuclear magnetic resonance spectroscopy (NMR) and mass spectroscopyconfirmed the identity of the synthesized compound. The purity of thecompound was analyzed by high performance liquid chromatography (HPLC)using a C-18 reverse phase column and water/acetonitrile as solvent(1–100% acetonitrile over 25 minutes).

Example 2 Synthesis of the 3-toluate of the morpholinepyridoxal-4,5-aminal(1-morpholino-1,3-dihydro-7-(p-toluoyloxy)-6-methylfuro(3,4-c)pyridine)

Anhydrous powdered potassium carbonate (5 g), acetone (100 mL), andmorpholine pyridoxal-4,5-aminal(1-morpholino-1,3-dihydro-7-hydroxy-6-methylfuro(3,4-c)pyridine) (1.11g, 5 mmoles) were mixed in a nitrogen-cooled, dry flask. The reactionmixture was cooled to between 0 and 5° C. and then p-toluoyl chloride(1.06 g, 6 mmoles) in acetone (20 mL) was added. This mixture wasstirred for two hours, followed by filtering out the solid andevaporating the solution to dryness under vacuum. The residue waschromatographed on silica gel using a mixture of ethyl acetate andhexane as solvent.

The purified solid was analyzed by thin layer chromatography (TLC), NMR,and mass spectroscopy. The purity of the synthesized compound wasconfirmed by HPLC as described in Example 1.

Example 3 Synthesis of the 3-toluate of pyridoxal(2-methyl-3-toluoyloxy-4-formyl-5-hydroxymethylpyridine)

Anhydrous potassium carbonate (10 g), acetone (100 mL), and pyridoxalhydrochloride (2.03 g, 10 mmoles) were mixed in a nitrogen-cooled, dryflask. The mixture was cooled to between 0 and 5° C. and then p-toluoylchloride (2.12 g, 12 mmoles) in acetone (20 mL) was added. The reactionmixture was stirred for two hours followed by filtering out the solidand evaporating the solution to dryness under vacuum. The residue waschromatographed on silica gel as described in Example 2.

The purified solid was analyzed by TLC, NMR, and mass spectroscopy. Thepurity of the compound was confirmed by HPLC as described in Example 1.

Alternative to the above-described method, the 3-toluate of pyridoxal issynthesized by reacting the compound of Example 2 with 80% aqueousacetic acid at 60° C. for 30 minutes, and then diluting with water andextracting by ethyl acetate. The ethyl acetate layer is washed with 5%aqueous sodium bicarbonate, dried with magnesium sulfate, and evaporatedto dryness. The compound is also analyzed as described supra.

Example 4 Synthesis of 3-β-naphthoate of the morpholinepyridoxal-4,5-aminal(1-morpholino-1,3-dihydro-7-(β-naphthoyloxy)-6-methylfuro(3,4-c)pyridine)

Anhydrous powdered potassium carbonate (5 g), acetone (100 mL), andmorpholine pyridoxal-4,5-aminal(1-morpholino-1,3-dihydro-7-hydroxy-6-methylfuro(3,4-c)pyridine) (1.11g, 5 mmoles) were mixed in a nitrogen-cooled, dry flask. The mixture wascooled to between 0 and 5° C. and then β-naphthoyl chloride (1.06 g, 6mmoles) in acetone (20 mL) was added. The reaction mixture was stirredfor two hours, and then the solid was filtered out and the solution wasevaporated to dryness under vacuum. The residue was chromatographedaccording to Example 2.

The purified solid was analyzed according to Example 2, and the puritywas confirmed according to Example 1.

Example 5 Synthesis of the 3-β-naphthoate of pyridoxal(2-methyl-3-β-naphthoyloxy-4-formyl-5-hydroxymethylpyridine)

Anhydrous potassium carbonate (10 g), acetone (100 mL), and pyridoxalhydrochloride (2.03 g, 10 mmoles) were mixed in a nitrogen-cooled, dryflask. The mixture was cooled to between 0 and 5° C. and thenβ-naphthoyl chloride (2.12 g, 12 mmoles) in acetone (20 mL) was addedand the mixture was stirred for two hours. The solid was filtered outand the solution was evaporated to dryness under vacuum. The residue waschromatographed according to Example 2.

The purified solid was analyzed according to Example 2, and the puritywas confirmed according to Example 1.

Alternative to the above-described synthesis, the 3-β-naphthoate ofpyridoxal is prepared by reacting the compound of Example 4 with 80%aqueous acetic acid at 60° C. for 30 minutes, followed by diluting withwater and extracting by ethyl acetate. The ethyl acetate layer is thenwashed with 5% aqueous sodium bicarbonate, dried with magnesium sulfate,and evaporated to dryness. The compound is also analyzed as describedsupra.

Example 6 Synthesis of 3-pivaloyl of the morpholine pyridoxal-4,5-aminal(1-morpholino-1,3-dihydro-7-pivaloyloxy)-6-methylfuro (3,4-c)pyridine)

Anhydrous powdered potassium carbonate (5 g), acetone (100 mL), andmorpholine pyridoxal-4,5-aminal(1-morpholino-1,3-dihydro-7-hydroxy-6-methylfuro(3,4-c)pyridine) (1.11μg, 5 mmoles) were mixed in a nitrogen-cooled, dry flask. The mixturewas cooled to between 0 and 5° C. and then pivaloyl chloride(trimethylacetyl chloride) (720 mg, 6 mmoles) in acetone (20 mL) wasadded. The reaction mixture was stirred for two hours. The solid wasthen filtered out and the solution was evaporated to dryness undervacuum. The residue was chromatographed according to Example 2.

The purified solid was analyzed according to Example 2, and the puritywas confirmed according to Example 1.

Example 7 Synthesis of 3-dimethylcarbamoyl of the morpholinepyridoxal-4,5-aminal(1-morpholino-1,3-dihydro-7-(dimethylcarbamoyloxy)-6-methylfuro(3,4-c)pyridine)

Anhydrous powdered potassium carbonate (5 g), acetone (100 mL), andmorpholine pyridoxal-4,5-aminal(1-morpholino-1,3-dihydro-7-hydroxy-6-methylfuro(3,4-c)pyridine) (1.11g, 5 mmoles) were mixed in a nitrogen-cooled, dry flask. The mixture wascooled to between 0 and 5° C. and then dimethylcarbamoyl chloride (642mg, 6 mmoles) in acetone (20 mL) was added. The reaction mixture wasstirred for two hours. The solid was then filtered out and the solutionwas evaporated to dryness under vacuum. The residue was chromatographedaccording to Example 2.

The purified solid was analyzed according to Example 2, and the puritywas confirmed according to Example 1.

Example 8 Synthesis of 3-acetylsalicyloyl of the morpholinepyridoxal-4,5-aminal(1-morpholino-1,3-dihydro-7-acetylsalicyloxy)-6-methylfuro(3,4-c)pyridine)

Anhydrous powdered potassium carbonate (5 g), acetone (100 mL), andmorpholine pyridoxal-4,5-aminal(1-morpholino-1,3-dihydro-7-hydroxy-6-methylfuro(3,4-c)pyridine) (1.11g, 5 mmoles) were mixed in a nitrogen-cooled, dry flask. The mixture wascooled to between 0 and 5° C. and then acetylsalicyloyl chloride (1.09g, 6 mmoles) in acetone (20 mL) was added. The reaction mixture wasstirred for two hours. The solid was then filtered out and the solutionwas evaporated to dryness under vacuum. The residue was chromatographedaccording to Example 2.

The purified solid was analyzed according to Example 2, and the puritywas confirmed according to Example 1.

Example 9 In Vitro—Ischemia Reperfusion in Isolated Rat Hearts andMeasurement of Left Ventricular Developed Pressure (LVDP)

Male Sprague-Dawley rats weighing 250–300 g are anaesthetized with amixture of ketamine (60 mg/kg) and xylazine (10 mg/kg). The hearts arerapidly excised, cannulated to a Langendorff apparatus and perfused withKrebs-Henseleit-solution, gassed with a mixture of 95% O₂ and 5% CO₂, pH7.4. The perfusate contained (in mM): 120 NaCl, 25 NaHCO₃, 11 glucose,4.7 KCl, 1.2 KH₂PO₄, 1.2 MgSO₄ and 1.25 CaCl₂.

The hearts are electrically stimulated at a rate of 300 beats/mim(Phipps and Bird Inc., Richmond, Va.) and a water-filled latex balloonis inserted in the left ventricle and connected to a pressure transducer(Model 1050BP; BYOPAC SYSTEM INC., Goleta, Calif.) for the leftventricular systolic measurements. The left ventricular end diastolicpressure (LVEDP) is adjusted at 10 mmHg at the beginning of theexperiment. In some experiments the left ventricular pressures aredifferentiated to estimate the rate of ventricular contraction (+dP/dt)and rate of ventricular relaxation (−dP/dt) using the Acknowledge 3.03software for Windows (BIOPAC SYSTEM INC.,) Goleta, Calif.). All heartsare stabilized for a period of 30 mim and then randomly distributed intonine different experimental groups (n=5–8 per group). The experimentalgroups are defined as follows:

-   -   1) Control group (control hearts are further perfused for 90        minutes for a total of 130 mim of continuous perfusion);    -   2) Ischemia reperfusion group (Ischemia reperfusion hearts are        made globally ischemic by stopping the coronary flow completely        for 30 mim and then the hearts are reperfused for 60 mim);    -   3) P-5-P (15 μM) treated group;    -   4) captopril (100 μM) treated group;    -   5) verapamil (0.01 μM)) treated group;    -   6) propranolol (3 mM) treated group;    -   7) PPADS (10 μM) treated group;    -   8) P-5-P+captopril treated group;    -   9) P-5-P+verapamil treated group;    -   10) P-5-P+propranolol treated group;    -   11) P-5-P+PPADS treated group.

Drug treatment is started 10 min before global ischemia followed by 30min global ischemia and 60 min reperfusion. At the end of someexperiments, the hearts are quickly freeze-clamped with a liquidnitrogen precooled Wollenberger tong. Rats are housed in clear cages ina temperature and humidity controlled room on a 12 hr light-dark cycle.Food and water are supplied ad libitum.

Hearts subject to 30 mim of ischemia followed by 60 mim of reperfusionshowed slight recovery in the contractile function as represented by29.5% recovery in LVDP (left ventricular developed pressure). Ascompared to the untreated group, treatment with P-5-P, captopril, orP-5-P and captopril showed better recoveries in LVDP by 78.2%, 61.4%,and 132% respectively (Table I).

TABLE I Effect of Pyridoxal-5-phosphate (P-5-P, 15 μM) and Captopril(100 μM) on % recovery of left ventricular systolic pressure (LVDP).LVDP LVEDP LVSP % recovery Drugs (B) (A) mmHg mmHg (LVDP) Unreated 87 ±7   25 ± 2.9 62 ± 5.6 87 ± 6.9 29.5 ± 3.7 P5P  80 ± 3.8 63 ± 5  35 ± 4.898 ± 8.2  78.2 ± 3.3* Captopril  78 ± 10.9  47 ± 8.6 54 ± 6.7 101 ± 14.6 61.4 ± 5.2* P5P +  89 ± 6.9  69 ± 7.4 28 ± 7.3 117 ± 8.4    132 ±7.5^(#) Captopril (A) = After ischemia, (B) = Before ischemia.

Hearts subject to 30 mim of ischemia followed by 60 min of reperfusionshowed slight recovery in the contractile function as represented by29.5% recovery in LVDP. As compared to the untreated group, treatmentwith P-5-P, verapamil, or P-5-P and verapamil showed better recoveriesin LVDP by 78.2%, 43%, and 109% respectively (Table II).

TABLE II Effect of Pyridoxal-5-phosphate (P-5-P, 15 μM) and Verapamil(0.01 μM) on % recovery of left ventricular systolic pressure (LVDP).LVDP LVEDP LVSP % recovery Drugs (B) (A) mmHg mmHg (LVDP) Untreated 87 ±7   25 ± 2.9 62 ± 5.6 87 ± 6.9 29.5 ± 3.7  P5P  80 ± 3.8 63 ± 5  35 ±4.8 98 ± 8.2 78.2 ± 3.3* Verapamil  54 ± 9.1  23 ± 4.5 55 ± 5.1 78 ± 7.7 43 ± 6.6 P5P +   78 ± 10.5   85 ± 11.7 34 ± 7.3 119 ± 8    109 ±4.6^(#) Verapamil (A) = After ischemia, (B) = Before ischemia.

Hearts subject to 30 mim of ischemia followed by 60 mim of reperfusionshowed slight recovery in the contractile function as represented by29.5% recovery in LVDP. As compared to the untreated group, treatmentwith P-5-P, PPADS, or P-5-P and PPADS showed better recoveries in LVDPby 78.2%, 61%, and 128% respectively (Table III).

TABLE III Effect of Pyridoxal-5-phosphate (P-5-P, 15 μM) and Pyridoxalphosphate 6-azophenyl-2'-4'disulfonic acid (PPADS 100 μM) on % recoveryof left ventricular systolic pressure (LVDP). LVDP LVEDP LVSP % recoveryDrugs (B) (A) mmHg mmHg (LVDP) Untreated 87 ± 7   25 ± 2.9 62 ± 5.6  87± 6.9 29.5 ± 3.7  P5P  80 ± 3.8 63 ± 5  35 ± 4.8  98 ± 8.2 78.2 ± 3.3*PPADS   92 ± 15.2   58 ± 13.6 57 ± 6.3  115 ± 11.5   61 ± 4.8* P5P +  82 ± 15.8  105 ± 22.8 34 ± 3.1  139 ± 21.6   128 ± 13.8# PPADS (A) =After ischemia, (B) = Before isehemia.

Hearts subject to 30 mim of ischemia followed by 60 mim of reperfusionshowed slight recovery in the contractile function as represented by29.5% recovery in LVDP. As compared to the untreated group, treatmentwith P-5-P, propranolol, or P-5-P and propranolol showed betterrecoveries in LVDP by 78.2%, 74%, and 120% respectively (Table IV).

TABLE IV Effect of Pyridoxal-5-phosphate (P-5-P, 15 μM) and Propranolol(3 μM) on % recovery of left ventricular systolic pressure (LVDP). LVDPLVEDP LVSP % recovery Drugs (B) (A) mmHg mmHg (LVDP) Untreated 87 ± 7  25 ± 2.9 62 ± 5.6  87 ± 6.9 29.5 ± 3.7  P5P  80 ± 3.8 63 ± 5  35 ± 4.8 98 ± 8.2 78.2 ± 3.3* Propranolol   61 ± 10.8  45 ± 9.7 27 ± 6.6   72 ±15.1   74 ± 4.9* P5P +   67 ± 12.6   75 ± 10.4 40 ± 4.2 115 ± 8.3    120± 15.5^(#) Propranolol (A) = After isehemia, (B) = Before ischemia

Tables I–IV demonstrate that P-5-P in addition to providing significantbenefit in ischemia reperfusion injury when given alone also improves oradds to the benefits associated with other commonly used drugs whengiven in combination with these drugs.

In addition to captopril, other angiotensin converting enzymeinhibitors, such as, for example, enalapril or imidapril, can similarlybe administered in place of captopril. In addition to verapamil, otherknown calcium channel blockers, such as, for example, nifedipine ordiltiazem, can similarly be administered in place of verapamil. Inaddition to propranolol, other β-adrenergic receptor antagonists suchas, for example, atenolol, timolol, and metoprolol can similarly beadministered in place of propranolol. Additionally, angiotensin IIreceptor antagonists such as, for example, losartan and valsartan can beused in the above example.

Example 10 In Vivo—Coronary Artery Ligation

Myocardial infarction is produced in male Sprague-Dawley rats (200–250g) by occlusion of the left coronary artery as described in Sethi etal., J. Cardiac Failure, 1(5) (1995) and Sethi et al., Am. J. Physiol.,272 (1997). The animals were anesthetized with ether, the skin incisedalong the left sternal border, the fourth rib cut proximal to thesternum, and retractors inserted. The pericardial sac was perforated andthe heart was exteriorized through the intercostal space. The leftcoronary artery was ligated about 2 mm from its origin with a 6–0 silksuture and the heart was repositioned in the chest. Throughout thecourse of the operation, the rats were maintained on a positive pressureventilation delivering a mixture of 95% O₂ and 5% CO₂ mixed with ether.

Rats are anesthetized with 1–5% isoflurane in 100% O₂ (2 L flow rate).The skin is incised along the left sterna border and the 4th rib is cutproximal to the sternum and a retractor inserted. The pericardial sac isopened and the heart externalized. The left anterior descending coronaryartery is ligated approximately 2 mm from its origin on the aorta usinga 6–0 silk suture. The heart is then repositioned in the chest and theincision closed via purse-string sutures.

Sham operated rats undergo identical treatment except that the artery isnot ligated. Mortality due to surgery is less than 1%. Unless indicatedin the text, the experimental animals showing infarct size >30% of theleft ventricle are used in this study. All animals are allowed torecover, allowed to receive food and water ad libitum, and aremaintained for a period of 21 days for Electrocardiogram (ECG),hemodynamic, and histological assessment.

Occlusion of the coronary artery in rats has been shown to producemyocardial cell damage which results in scar formation in the leftventricle and heart dysfunction. While the complete healing of the scaroccurs within 3 weeks of the coronary occlusion, mild, moderate andsevere stages of congestive heart failure have been reported to occur at4, 8 and 16 weeks after ligation. Accordingly, the contractiledysfunction seen at 3 weeks after the coronary occlusion in rats is dueto acute ischemic changes.

The rats are housed in clear cages in a temperature and humiditycontrolled room, on a 12 hour light-dark cycle. Food and water aresupplied ad libitum. After surgery, rats are randomly assigned totreatment or non-treatment in both sham and experimental groups.Randomization of animals was performed and treatment begins 1 hour aftercoronary occlusion and continues for 21 days. The total duration ofexperiments in each case is 21 days. The groups are as follows:

-   1) sham operated;-   2) coronary artery ligated (treatment with equal volumes of saline);-   3) coronary artery ligated (treated with 10 mg/kg P-5-P);-   4) coronary artery ligated (treated with 100 mg/kg captopril);-   5) coronary artery ligated (treated with 50 mg/kg propranolol);-   6) coronary artery ligated (treated with 100 mg/kg aspirin);-   7) coronary artery ligated (treated with 25 mg/kg verapamil);-   8) coronary artery ligated (treated with 10 mg/kg P-5-P+100 mg/kg    captopril);-   9) coronary artery ligated (treated with 10 mg/kg P-5-P+50 mg/kg    propranolol);-   10) coronary artery ligated (treated with 10 mg/kg P-5-P+100 mg/kg    aspirin);-   11) coronary artery ligated (treated with 10 mg/kg P-5-P+25 mg/kg    verapamil).

P-5-P (10 mg/kg), captopril (100 mg/kg), propranolol (50 mg/kg),verapamil (25 mg/kg) and aspirin (100 mg/kg) were administered oncedaily by gastric tube.

Acute myocardial infarction resulted in a total mortality of 35% % inthe untreated group of rats in 21 days. The highest mortality occurredwithin the first 2 days following occlusion. As compared to theuntreated group, treatment with P-5-P, aspirin, or P-5-P and aspirinshowed lower mortality rates of 15%, 25%, 15%, respectively (FIG. 1).

Acute myocardial infarction resulted in a total mortality of 35% % inthe untreated group of rats in 21 days. The highest mortality occurredwithin the first 2 days following occlusion. As compared to theuntreated group, treatment with P-5-P, captopril, or P-5-P and captoprilshowed lower mortality rates of 10%, 15%, 20%, respectively (FIG. 2).

Acute myocardial infarction resulted in a total mortality of 35% % inthe untreated group of rats in 21 days. The highest mortality occurredwithin the first 2 days following occlusion. As compared to theuntreated group, treatment with P-5-P, propranolol, or P-5-P andpropranolol showed lower mortality rates of 15%, 20%, 20%, respectively(FIG. 3).

Acute myocardial infarction resulted in a total mortality of 35% % inthe untreated group of rats in 21 days. The highest mortality occurredwithin the first 2 days following occlusion. As compared to theuntreated group, treatment with P-5-P, verapamil, or P-5-P and verapamilshowed lower mortality rates of 15%, 25%, 10%, respectively (FIG. 4).

In addition to captopril, other angiotensin converting enzymeinhibitors, such as, for example, enalapril or imidapril, can similarlybe administered in place of captopril. In addition to verapamil, otherknown calcium channel blockers, such as, for example, nifedipine ordiltiazem, can similarly be administered in place of verapamil. Inaddition to propranolol, other β-adrenergic receptor antagonists suchas, for example, atenolol, timolol, and metoprolol can similarly beadministered in place of propranolol. In addition to aspirin, otherantithrombolytic agents such as, for example, antiplatelet agents andheparin can similarly be administered in place of aspirin. Additionally,angiotensin II receptor antagonists such as, for example, losartan andvalsartan can be used in the above example.

These animals are used in Examples 11 and 12 below. For EKG studies,these animals are used as their controls before surgery, so that beforesurgery is done on these animals EKG traces are taken which are thenused as controls for the same animals after surgery.

Example 11 In Vivo—Hemodynamic Changes

The animals are prepared and grouped as described in Example 10 and wereanesthetized with a solution of ketamine/xylazine which was injected. Tomaintain adequate ventilation, the trachea was intubated; the rightcarotid artery was exposed for introducing a microtip pressuretransducer (model SPR-249, Millar, Houston, Tex.) into the leftventricle. The catheter was secured with a silk ligature around theartery, and various hemodynamic parameters such as left ventricularsystolic pressure (LVSP), left ventricular end diastolic pressure(LVEDP), rate of contraction (+dp/dt), rate of relaxation (−dP/dt) wererecorded and calculated on a computer system using a Acknowledge 3.1software.

Once the hemodynamic parameters were measured the animals weresacrificed and hearts removed for measurement of heart weight, rightventricular weight, left ventricular weight and scar weight. Becausecomplete healing of the scar in rats after coronary occlusion requiresapproximately 3 weeks, scar weight were measured only at 21 days.

FIGS. 5–8 demonstrate that the occlusion of coronary artery in rats for21 days produces a significant scar evident by scar weight. Furthermore,FIGS. 5–8 demonstrate that P-5-P has a significant beneficial effect onscar weight in groups where P-5-P treatment is either given alone or incombination with verapamil, aspirin, captopril, or propranolol,respectively.

FIGS. 9–12 demonstrate that P-5-P has a significant beneficial effect onrate of contraction (+dP/dt) in groups where P-5-P treatment is eithergiven alone or in combination with verapamil, aspirin, captopril, orpropranolol, respectively.

FIGS. 13–16 demonstrate that P-5-P has a significant beneficial effecton rate of relaxzation (+dP/dt) in groups where P-5-P treatment iseither given alone or in combination with verapamil, aspirin, captopril,or propranolol, respectively.

FIGS. 17–20 demonstrate that P-5-P has a significant beneficial effecton rate of left ventricular end diastolic pressure (LVEDP) in groupswhere P-5-P treatment is either given alone or in combination withverapamil, aspirin, captopril, or propranolol, respectively.

FIGS. 21–24 demonstrate that P-5-P has a significant beneficial effecton whole heart weight in groups where P-5-P treatment is either givenalone or in combination with verapamil, aspirin, captopril, orpropranolol, respectively.

FIGS. 25–28 demonstrate that P-5-P has a significant beneficial effecton right ventricular weight in groups where P-5-P treatment is eithergiven alone or in combination with verapamil, aspirin, captopril, orpropranolol, respectively.

In addition to captopril, other angiotensin converting enzymeinhibitors, such as, for example, enalapril or imidapril, can similarlybe administered in place of captopril. In addition to verapamil, otherknown calcium channel blockers, such as, for example, nifedipine ordiltiazem, can similarly be administered in place of verapamil. Inaddition to propranolol, other β-adrenergic receptor antagonists suchas, for example, atenolol, timolol, and metoprolol can similarly beadministered in place of propranolol. In addition to aspirin, otherantithrombolytic agents such as, for example, antiplatelet agents andheparin can similarly be administered in place of aspirin. Additionally,angiotensin II receptor antagonists such as, for example, losartan andvalsartan can be used in the above example.

Example 12 In Vivo—Hypertension

It has been well demonstrated by various investigators that feeding8–10% sucrose in water induces hypertension in rats. Zein et al., intheir publication entitled Sugar-Induced Blood Pressure Elevations Overthe Lifespan of Three Substrains of Wistar Rats in Am. Coil. Nutr., 17(1), 36–37, 1998, have shown that high dietary sucrose can chronicallyincrease systolic blood pressure (SBP) in three substrains of Wistarrats. Increased concentrations of circulating insulin were found inWistar Kyoto rats and Munich Wistar rats suggesting that theglucose/insulin system was involved, at least in these two substrains,in the maintenance of high SBP levels during chronic, heavy sugaringestion. Hulman et al. in their publication entitled The Effect ofExcess Dietary Sucrose on Growth, Blood Pressure, and Metabolism inDeveloping Sprague-Dawley Rats in Pediatr. Res., 36:95–101, have shownthat consumption of a diet in which complex carbohydrate has beenreplaced by sucrose causes both elevated blood pressure and insulinresistance in juvenile rats with no genetic predisposition in eithercondition. Although blood pressure increased with increasing age andbody weight in all four diet groups, higher blood pressures were clearlycorrelated with the sucrose diet in both males and females. The higherblood pressure cannot be explained by greater total body weight in thesucrose-fed animals, because there was no difference in weight betweencontrol-fed and sucrose-fed rats in each sex group. Reaven et al., intheir publication entitled Sugar-Induced Hypertension in Sprague-DawleyRats in Am. J. Hypertens; 1991:610–614, have shown that the ability ofsimple sugars to increase plasma insulin and TG concentration and raiseblood pressure is not unique to fructose, but can also be seen whenSprague-Dawley rats eat diets enriched with either glucose or sucrose.Since plasma glucose concentrations did not change, it is assumed thatglucose-fed and sucrose-fed Sprague-Dawley rats also became moreresistant to insulin-stimulated glucose uptake. In applying this model,the concurrent administration of pyridoxal-5′-phosphate and captopril orverapamil significantly decreases the sucrose-induced increase insystolic blood pressure (SBP).

The blood pressure is monitored using the tail cuff method. The SBP isdetected on an amplifier and the Acknowledge™ computer software programis used to determine the calculations.

The effect of concurrent administration of pyridoxal-5′-phosphate andcaptopril or verapamil on systolic blood pressure (marker ofhypertension) in 10% sucrose induced hypertension in rats is determined.

FIGS. 29A and 9B demonstrate that P-5-P has a significant beneficialeffect on systolic blood pressure in groups where P-5-P treatment iseither given alone or in combination with verapamil or captopril 1 weekprior to inducing hypertension in rats with a sucrose diet.

FIGS. 29A and 29B demonstrate that P-5-P has a significant beneficialeffect on systolic blood pressure in groups where P-5-P treatment iseither given alone or in combination with verapamil or captopril 1 weekprior to inducing hypertension in rats with a sucrose diet.

FIGS. 30A and 30B demonstrate that P-5-P has a significant beneficialeffect on systolic blood pressure in groups where P-5-P treatment iseither given alone or in combination with verapamil or captopril thesame day as inducing hypertension in rats with a sucrose diet.

FIGS. 31A and 31B demonstrate that P-5-P has a significant beneficialeffect on systolic blood pressure in groups where P-5-P treatment iseither given alone or in combination with verapamil or captopril twoweeks after inducing hypertension in rats with a sucrose diet.

In addition to captopril, other angiotensin converting enzymeinhibitors, such as, for example, enalapril or imidapril, can similarlybe administered in place of captopril. In addition to verapamil, otherknown calcium channel blockers, such as, for example, nifedipine ordiltiazem, can similarly be administered in place of verapamil. Inaddition to propranolol, other β-adrenergic receptor antagonists suchas, for example, atenolol, timolol, and metoprolol can similarly beadministered in place of propranolol. Additionally, angiotensin IIreceptor antagonists such as, for example, losartan and valsartan can beused in the above example.

It should be noted that, as used in this specification and the appendedclaims, the singular forms “a,” “an,” and “the” include plural referentsunless the content clearly dictates otherwise. Thus, for example,reference to a composition containing “a compound” includes a mixture oftwo or more compounds.

Although embodiments of the invention have been described above, it isnot limited thereto, and it will be apparent to persons skilled in theart that numerous modifications and variations form part of the presentinvention insofar as they do not depart from the spirit, nature, andscope of the claimed and described invention.

1. A method of treating ischemia reperfusion injury in a mammal comprising: concurrently administering to the mammal a therapeutically effective amount for treating ischemia reperfusion injury of a combination of a compound selected from the group consisting of pyridoxal-5′-phosphate, pyridoxal, pyridoxamine, a 3-acylated pyridoxal analogue, a pharmaceutically acceptable acid addition salt thereof, and a mixture thereof, and a therapeutic cardiovascular compound selected from the group consisting of an angiotensin converting enzyme inhibitor, an angiotensin II receptor antagonist, a calcium channel blocker, a β-adrenergic receptor antagonist, a vasodilator, a diuretic, an α-adrenergic receptor antagonist, an antioxidant, and a mixture thereof, wherein the 3-acylated pyridoxal analogue is a compound of the formula

wherein R₁ is a straight or branched alkyl group, a straight or branched alkenyl group, in which an alkyl or alkenyl group may be interrupted by a nitrogen or oxygen atom; an alkoxy group; a dialkylamino group; or an unsubstituted or substituted aryl group; and R₂ is a secondary amino group.
 2. A method according to claim 1, wherein the 3-acylated pyridoxal analogue is a compound of the formula

wherein R₁ is a straight or branched alkyl group, a straight or branched alkenyl group, in which an alkyl or alkenyl group may be interrupted by a nitrogen or oxygen atom; an alkoxy group; a dialkylamino group; or an unsubstituted or substituted aryl group.
 3. A method according to claim 1, wherein the 3-acylated pyridoxal analogue is a compound of the formula

wherein R₁ is a straight or branched alkyl group, a straight or branched alkenyl group, in which an alkyl or alkenyl group may be interrupted by a nitrogen or oxygen atom; an alkoxy group; a dialkylamino group; or an unsubstituted or substituted aryl group; and R₂ is a secondary amino group.
 4. A method according to claim 1, wherein the angiotensin converting enzyme inhibitor is captopril, enalapril, lisinopril, benazapril, fosinopril, quinapril, ramipril, spirapril, imidapril, or moexipril.
 5. A method according to claim 1, wherein the angiotensin II receptor antagonist is losartan or valsartan.
 6. A method according to claim 1, wherein the calcium channel blocker is verapamil, diltiazem, nicardipine, nifedipine, amlodipine, felodipine, nimodipine, or bepridil.
 7. A method according to claim 1, wherein the compound is administered enterally or parenterally and the therapeutic cardiovascular compound is administered enterally or parenterally.
 8. A method according to claim 1, wherein the compound and the therapeutic cardiovascular compound are administered in a single dosage form.
 9. A method according claim 1, wherein the vasodilator is hydralazine, nitroglycerin, or isosorbide dinitrate.
 10. A method according to claim 1, wherein the α-adrenergic receptor antagonist is prazosin, doxazocin, or labetalol.
 11. A method according to claims 1, wherein the antioxidant is vitamin E, vitamin C, or an isoflavone.
 12. A method according to claim 1, wherein the diuretic is furosemide, diuril, amiloride, or hydrodiuril.
 13. A method according to claim 1, wherein the α-adrenergic receptor antagonist is atenolol, propranolol, timolol, or metoprolol. 